Cancer is the second most common cause of death in the United States, exceeded only by heart disease. In the United States, cancer accounts for 1 of every 4 deaths. The 5-year relative survival rate for all cancer patients diagnosed in 1996-2003 is 66%, up from 50% in 1975-1977 (Cancer Facts & Figures American Cancer Society: Atlanta, Ga. (2008)). The rate of new cancer cases decreased by an average 0.6% per year among men between 2000 and 2009 and stayed the same for women. From 2000 through 2009, death rates from all cancers combined decreased on average 1.8% per year among men and 1.4% per year among women. This improvement in survival reflects progress in diagnosing at an earlier stage and improvements in treatment. Discovering highly effective anticancer agents with low toxicity is a primary goal of cancer research.
Microtubules are cytoskeletal filaments consisting of αβ-tubulin heterodimers and are involved in a wide range of cellular functions, including shape maintenance, vesicle transport, cell motility, and division. Tubulin is the major structural component of the microtubules and a well verified target for a variety of highly successful anti-cancer drugs. Compounds that are able to interfere with microtubule-tubulin equilibrium in cells are effective in the treatment of cancers. Anticancer drugs like taxol and vinblastine that are able to interfere with microtubule-tubulin equilibrium in cells are extensively used in cancer chemotherapy.
Unfortunately, microtubule-interacting anticancer drugs in clinical use share two major problems, resistance and neurotoxicity.
Malignant melanoma is the most dangerous form of skin cancer, accounting for about 75% of skin cancer deaths. The incidence of melanoma is rising steadily in Western populations. The number of cases has doubled in the past 20 years. Around 160,000 new cases of melanoma are diagnosed worldwide each year, and it is more frequent in males and Caucasians. According to a WHO Report, about 48,000 melanoma-related deaths occur worldwide per year.
Currently there is no effective way to treat advanced/metastatic melanoma. It is highly resistant to current chemotherapy, radiotherapy, and immunotherapy. Advanced/metastatic melanoma has a very poor prognosis, with a median survival rate of 6 months and a 5-year survival rate of less than 5%.
Various chemotherapy agents are used, including dacarbazine (also termed DTIC), immunotherapy (with interleukin-2 (IL-2) or interferon (IFN)), as well as local perfusion, are used by different centers. The overall success in metastatic melanoma is quite limited. IL-2 (Proleukin) is the first new therapy approved for the treatment of metastatic melanoma in 20 years. However, it provides only less than 5% of complete remission in patients. In recent years, great efforts have been attempted in fighting advanced melanoma. Neither combinations of DTIC with other chemotherapy drugs (e.g., cisplatin, vinblastine, and carmustine) nor adding interferon-α2b to DTIC have shown a survival advantage over DTIC treatment alone. Most recently, clinical trials with antibodies and vaccines to treat advanced melanoma also failed to demonstrate satisfactory efficacy. Ipilimumab (Yervoy®) is a drug that uses your immune system to fight melanoma. Ipilimumab is used to treat advanced melanoma that has spread beyond its original location. Targeted therapy uses medications designed to target specific vulnerabilities in cancer cells.
The discovery of the BRAF mutation in ˜60% of melanoma patients and the FDA approved BRAF inhibitors (BRAFi; e.g. vemurafenib and dabrafenib (GSK2118436)) and a MEK inhibitor (MEKi; e.g. trametinib (GSK1120212), RO5068760) have shown impressive clinical responses in the treatment of BRAFV600 mutant melanomas. The upfront use of BRAFi+MEKi combination is highly effective during initial therapy, but due to tumor heterogeneity and activations of alternative pathways, resistance develops within ˜9 months leading to recurrent disease and death of patients.
Vemurafenib (Zelboraf®) is a targeted therapy approved to treat advanced melanoma that cannot be treated with surgery or melanoma that has spread through the body. With regard to melanoma, vemurafenib only treats tumors that have a certain genetic mutation (BRAFV600). Likewise, vemurafenib and other BRAF inhibitors may be active in a variety of BRAF mutant cancers. Examples in which B-RAF is mutated at a high frequency include melanoma (30-60%), thyroid cancer (30-50%), colorectal cancer (5-20%), ovarian cancer (˜30%), and other cancers (1-3%) (Wellbrock C, Karasarides M, Marais R. “The Raf Protein Takes Centre Stage”. Nat. Rev. (2004) 5: 875-885).
The sustained clinical activity of vemurafenib in patients with BRAFV600 mutant melanoma is limited by the rapid development of acquired resistance (Lee J T, Li L, Brafford P A, et al. “PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas.” Pigment Cell Melanoma Res. (2010) 23: 820-827; Yang H, Higgins B, Kolinsky K, et al. “RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models”. Cancer Res. (2010) 70: 5518-5527; Yang H, Higgins B, Kolinsky K, et al. “Antitumor activity of BRAF inhibitor vemurafenib in preclinical models of BRAF-mutant colorectal cancer”. Cancer Res. (2012) 72: 779-789.). The mechanisms of resistance development have been widely investigated (Little A S, Smith P D, Cook S J. “Mechanisms of acquired resistance to ERK1/2 pathway inhibitors”. Oncogene (2013) 32(10): 1207-1215; Bollag G, Hirth P, Tsai J, et al. “Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma”. Nature (2010) 467: 596-599; Flaherty K T. “Targeting metastatic melanoma”. Annu Rev Med. (2012) 63: 171-183; Su F, Bradley W D, Wang Q, et al. “Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation”. Cancer Res. (2012) 72: 969-978.). Many different mechanisms have been proposed in the literature, including intrinsic resistance to BRAFi, the amplification of the BRAF oncogene (Shi H, Moriceau G, Kong X, et al. “Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance.” Nat. Commun. (2012) 3: 724), up-regulation or activating mutations of downstream MEK kinases, the upregulation of CRAF expression (Montagut C, Sharma S V, Shioda T, et al. “Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma”. Cancer Res. (2008) 68: 4853-4861), oncogenic activation of NRAS (Nazarian R, Shi H, Wang Q, et al. “Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation”. Nature (2010) 468: 973-977), up-regulated EGFR-SFK-STAT3 pathway (Girotti M R, Pedersen M, Sanchez-Laorden B, et al. “Inhibiting EFG receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma.” Cancer Discov. (2013) 3(2): 158-167), gatekeeper mutations (Whittaker S, Kirk R, Hayward R, et al. “Gatekeeper mutations mediate resistance to BRAF-targeted therapies.” Sci. Transl. Med. (2010) 2: 35ra41; Balzano D, Santaguida S, Musacchio A, Villa F. “A general framework for inhibitor resistance in protein kinases.” Chem. Biol. (2011) 18: 966-975; Sierra J R, Cepero V, Giordano S. “Molecular mechanisms of acquired resistance to tyrosine kinase targeted therapy.” Mol. Cancer (2010) 9: 75), upregulation of growth factor receptors such as insulin-like growth factor 1 receptor (IFG1R)(Villanueva J, Vultur A, Lee J T, et al. “Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K”. Cancer Cell (2010) 18: 683-695) or platelet-derived growth factor receptor (PDGFR), and several other resistance mechanisms (Wilson T R, Fridlyand J, Yan Y, et al. “Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors”. Nature (2012) 487: 505-509; Straussman R, Morikawa T, Shee K, et al. “Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion”. Nature (2012) 487: 500-504). Several methods to maintain phosphorylated extracellular-signal-related kinase 1 and 2 (p-ERK1/2) levels in the presence of BRAF inhibitor drugs have been described, including ERKkinase 1 (MEK1) mutation, recruitment of alternative MEK1/2 activators, RAS mutation or up-regulation of receptor tyrosine kinases (RTKs). Thus in many cases, vemurafenib-resistant cells are cross-resistant to MEK inhibitors (Little A S, Smith P D, Cook S J. “Mechanisms of acquired resistance to ERK1/2 pathway inhibitors”. Oncogene (2013) 32(10): 1207-1215; Atefi M, von Euw E, Attar N, et al. “Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway.” PLoS One (2011) 6: e28973; Poulikakos P I, Persaud Y, Janakiraman M, et al. “RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)” Nature (2011) 480: 387-390). Because one of the major acquired vemurafenib-resistant mechanisms is sustained downstream MEK/ERK activation, the combination of BRAFi+MEKi that target elements within the RAF-MEK-ERK pathway has attracted the most attention leading to FDA approval of dabrafenib+trametinib combination in 2013. However, due to tumor heterogeneity and activations of alternative pathways in melanoma, resistance to this combination treatment develops in an average of 9.4 months, and it has little clinical activity once resistance develops.
Drug combination using agents with distinct anti-cancer mechanisms can enhance tumor response and patient survival, especially in the treatment of advanced cancer patients (Carrick S, Parker S, Wilcken N, et al. “Single agent versus combination chemotherapy for metastatic breast cancer”. Cochrane Database Syst. Rev. 2005: CD003372; Fassnacht M, Terzolo M, Allolio B, et al. “Combination chemotherapy in advanced adrenocortical carcinoma”. N. Engl. J. Med. (2012) 366: 2189-2197; Pannu V, Karna P, Sajja H K, et al. “Synergistic antimicrotubule therapy for prostate cancer”. Biochem. Pharmacol. (2011) 81: 478-487). Although the combinations of vemurafenib with agents targeting the same mitogen-activated protein kinase (MAPK) pathway such as MEK or ERK inhibitors have been extensively investigated and have shown clinical efficacy (Greger J G, Eastman S D, Zhang V, et al. “Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations” Mol. Cancer Ther. (2012) 11: 909-920; Patel S P, Lazar A J, Papadopoulos N E, et al. “Clinical responses to selumetinib (AZD6244; ARRY-142886)-based combination therapy stratified by gene mutations in patients with metastatic melanoma”. Cancer (2013) 119(4): 799-805; Flaherty K T, Infante J R, Daud A, et al. “Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations”. N. Engl. J. Med. (2012) 367: 1694-1703), they can only arrest cells in the G0/G1 phase. Such combination strategies are unlikely effective in dealing with resistant cells that can escape from this cell cycle arrest.
Chronically selected vemurafenib-resistant human melanoma cells (e.g., A375RF21) could not be blocked on the G0/G1 phase by vemurafenib at the effective concentration to sensitive parental cell line (i.e., A375), and the vemurafenib-resistant cells readily progressed into the G2/M phase (FIG. 2A). Thus, a combination of vemurafenib with a compound that strongly induces subsequent G2/M phase block should successfully capture the vemurafenib resistant cells leaking from G0/G1 arrest, thus produce strong synergy.
Recently, a novel class of anti-mitotic agents, represented by the scaffold of 2-aryl-4-benzoyl-imidazoles (ABIs) has been discovered (Chen J, Li C M, Wang J, et al. “Synthesis and antiproliferative activity of novel 2-aryl-4-benzoyl-imidazole derivatives targeting tubulin polymerization”. Bioorg. Med. Chem. (2011) 19: 4782-4795; Chen J, Wang Z, Li C M, et al. “Discovery of novel 2-aryl-4-benzoyl-imidazoles targeting the colchicines binding site in tubulin as potential anticancer agents”. J. Med. Chem. (2010) 53: 7414-7427; Chen J, Ahn S, Wang J, et al. “Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents”. J. Med. Chem. (2012) 55: 7285-7289; Li C M, Lu Y, Chen J, et al. “Orally bioavailable tubulin antagonists for paclitaxel-refractory cancer”. Pharm. Res. (2012) 29: 3053-3063). These compounds presented anti-proliferation IC50 values at the low nanomolar (nM) range in several human and mouse melanoma cell lines. They bind to tubulin at colchicine binding site. Compared with many existing tubulin inhibitors such as paclitaxel and vinblastine, ABI compounds can effectively circumvent several clinically relevant multidrug resistant mechanisms, including drug resistance mediated by P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), and breast cancer resistant proteins (BCRP). In vivo study indicated that they significantly inhibited melanoma B16-F10 cell lung metastasis in mice (Wang Z, Chen J, Wang J, et al. “Novel tubulin polymerization inhibitors overcome multidrug resistance and reduce melanoma metastasis to the lung”. Pharm. Res. (2012) 29: 3040-3052).
With the rapidly rising incidence of cancer, and especially melanoma, and the high resistance to current therapeutic agents, identifying more efficacious drug combinations targeting alternative pathways to overcome BRAFi-resistance in melanoma will significantly benefit patients. In addition, because BRAF mutations are also common in many other types of cancers including ovarian, colorectal, and papillary thyroid cancers. Developing novel combination strategies may have a broader impact for these types of cancers where the use of existing BRAFi+MEKi combinations show little clinical activity, and are urgently needed.